CN108771659B - Preparation method of indocyanine green fluorescent-labeled trans-anethole-albumin nanoparticles - Google Patents
Preparation method of indocyanine green fluorescent-labeled trans-anethole-albumin nanoparticles Download PDFInfo
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Classifications
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- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
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- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
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- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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- B82—NANOTECHNOLOGY
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Abstract
The invention relates to a preparation method of a trans-anethole-albumin nano particle with a green indocyanine fluorescent tracer; by using albumin as a raw material and by adjusting the proportion and the solvent of the encapsulated green indocyanine fluorescent dye and the trans-anethole drug, the particle size of the particles is fully controlled, and particles with the particle size of 20-50 nanometers are prepared; the green indocyanine fluorescent dye and the trans-anethole are simultaneously encapsulated in the albumin nanoparticles, so that the albumin nanoparticles have the functions of treatment and imaging. The in vitro drug release is determined by high performance liquid chromatography, the release rate of the sustained release 96h in phosphate buffer solution only reaches 31 percent, and the sustained release effect is obvious. The invention has simple and convenient synthesis process, no toxicity, rapidness and large yield, and obviously reduces the grain diameter of the granules compared with the prior art. In an in vitro slow release test, the trans-anethole-albumin nano-particles labeled by green indocyanine green fluorescence have a good slow release effect, the release degree in 96 hours only reaches 31%, and the slow release effect is achieved.
Description
Technical Field
The invention relates to the technical field of nano medicines, and particularly relates to a preparation method of a trans-anethole-albumin nano particle labeled by green indole phthalocyanine in a fluorescent manner.
Background
Trans-anethole is the main component of anise oil, is a natural antioxidant, and has antibacterial, antifungal and anesthetic properties. Studies show that anethole can effectively control some non-immune acute inflammation related diseases through mechanisms such as TNF signal inhibition, inhibition on generation and/or release of PGE 2 and NO and the like, and shows anti-inflammatory properties. Trans-anethole is found to reduce the concentration of cytokines such as TNF-alpha and the like in an acute and persistent inflammation model of a mouse; trans-anethole also exhibits inhibitory effects on total protein concentration, inflammatory cells, inflammatory mediators and the like in Lipopolysaccharide (LPS) -induced acute lung inflammation models.
Although trans-anethole has a good anti-inflammatory effect, its extremely strong hydrophobicity greatly limits the direct application of trans-anethole in vivo. In the current experiment, the compound can only be dissolved in organic solvents such as dimethyl sulfoxide, dichloromethane, tween-20 and the like for use, and the organic solvents have certain toxicity and low solubility and cannot be used for large-dose administration. Studies have attempted to use less toxic PEG polymers as co-solvents for trans-anethole, but with more than 10% PEG, mice develop hemolysis. Meanwhile, trans-anethole is usually administrated by lung irrigation in the treatment of pneumonia, so that the process is painful, multiple times of administration are needed, and great pain is brought to experimental animals.
In order to overcome the defects of hydrophobic drugs, reduce the toxic and side effects of organic solvents and reduce the administration frequency, people choose to adopt nano-drugs to solve the problems. The nano medicament refers to nano drug particles with the particle size of 10-1000 nanometers, and can wrap drugs or other functional molecules in the nano particles or modify the surfaces of the nano particles and transport the nano particles to a target site. The nano-particles have the advantages of high drug loading, simple preparation method, low toxicity, capability of circulating in vivo for a long time and slowly releasing drugs, and the like. However, no research report about the trans-anethole nano-particles exists at present.
The albumin nano-particles are green, degradable and good in safety, and can be used for loading fat-soluble medicines. Stirring and mixing the albumin water solution and the oily solvent for dissolving the medicine at a high speed to form emulsion, then adding glutaraldehyde for crosslinking, and finally performing rotary evaporation to remove the oil phase to obtain the stable nanoparticles. The method can continuously produce the albumin nano-particles carrying the medicine in large quantity. The prepared albumin nano-particles have the characteristics of biodegradability, easy preparation, good repeatability and the like.
In order to endow the nanoparticle with a tracing effect and verify that the nanoparticle can be phagocytized by macrophages to play a verification and regulation role, organic dyes can be added in the preparation process of the particle to perform fluorescence imaging. Therefore, the medicine particles which have the advantages of uniform particles, good balling property, avoidance of toxicity of organic solvents, good slow release effect, effective improvement of the use efficiency of the trans-anethole medicine, enhancement of the treatment effect, real-time tracking of nano particles and the like are developed, and the medicine particles have great significance and have important scientific research and clinical application prospects in the technical fields of biotechnology and medicine.
Disclosure of Invention
The invention provides a preparation method of novel albumin particles, which has the advantages of small particle size, small cytotoxicity, relatively high drug loading capacity, real-time tracing, nano particles and the like.
The technical scheme of the invention comprises the following steps: by using albumin as a raw material and by adjusting the proportion and the solvent of the encapsulated green indocyanine fluorescent dye and the trans-anethole drug, the particle size of the particles is fully controlled, and particles with the particle size of 20-50 nanometers are prepared; the green indocyanine fluorescent dye and the trans-anethole are simultaneously encapsulated in the albumin nanoparticles, so that the albumin nanoparticles have the functions of treatment and imaging. The in vitro drug release is measured by high performance liquid chromatography, and the release rate of the sustained release tablet in phosphate buffer (PH 7.4) for 96 hours only reaches 31 percent, thereby having obvious sustained release effect.
The concrete description is as follows:
a preparation method of indocyanine green fluorescent-labeled trans-anethole-albumin nanoparticles; the method comprises the following steps:
1) preparing drug-loaded albumin nanoparticles: preparing 2-2.5 ml of 10-20 mg/ml albumin aqueous solution;
2) adding 40-60 mg/ml prepared by dichloromethane, ethanol or dimethyl sulfoxide, 10-15 mul of trans-anethole solution and 1-3 mul of 80-100 mg/ml indocyanine green solution;
3) after stirring, adding 70-100 mul of glutaraldehyde crosslinking agent aqueous solution, and stirring for reaction at 5-50 ℃;
4) after the reaction is finished, filtering by using a water system filter membrane;
5) and (4) centrifugally separating the filtrate, and washing with deionized water to obtain the indocyanine green fluorescent-labeled trans-anethole-albumin nano-particles.
Preferably, the step 3) is carried out for stirring reaction for 2-6 hours at the temperature of 5-50 ℃ and at the speed of 600-800 rpm/min.
Preferably, step 4) is performed by filtration through a 220nm aqueous membrane.
Preferably, the filtrate obtained in the step 5) is centrifuged at 6000-8,000 rpm/min for 3-6 minutes.
Preferably, step 5) deionized water washing is carried out for 3-6 times.
The trans-anethole and green indole phthalocyanine encapsulated albumin nanoparticles prepared by the method have the diameter of 20-50 nanometers and the drug loading rate of 5-8%.
Preferably, the albumin is bovine serum albumin or human serum albumin.
The in vitro release rule of the trans-anethole-bovine serum albumin nanoparticles is examined by adopting high performance liquid chromatography for the in vitro release of the particles, phosphate buffer is used as a release medium, stock solution is divided into a plurality of parts, and meanwhile, the particles are shaken on a shaking table at a constant speed of 37 ℃, sampled at regular time and the release rate is calculated.
The invention has the advantages that: 1) the synthesis process is simple, convenient and fast, and has no toxicity and no pollution. 2) The diameter of the indocyanine green trans-anethole-albumin nano-particles prepared by the invention is 20-50 nanometers, the drug loading rate is 5-8%, and the cell survival rate is 80-95%. Compared with the prior art, the drug loading is improved by 4 percent, the particle size is reduced by hundreds of nanometers, and the application in cells and animal bodies is more convenient. 3) The particles prepared by the method can track the positions of the particles in real time by observing the distribution condition of the green indocyanine fluorescent dye in the nano-carrier cells. 4) The in vitro drug release is measured by High Performance Liquid Chromatography (HPLC), and the release rate in PBS with pH 6.5 reaches 31 percent within 96 hours, which indicates that the particle has the sustained-release effect.
Drawings
FIG. 1-1: example 1 transmission electron microscopy photographs of indocyanine green fluorescently tagged trans-anethole-albumin nanoparticles were prepared.
FIGS. 1-2: example 2 transmission electron microscopy photographs of indocyanine green fluorescently tagged trans-anethole-albumin nanoparticles were prepared.
FIGS. 1 to 3: example 3 transmission electron microscopy photographs of indocyanine green fluorescently tagged trans-anethole-albumin nanoparticles were prepared.
FIGS. 1 to 4: example 4 transmission electron microscopy photographs of indocyanine green fluorescently tagged trans-anethole-albumin nanoparticles were prepared.
FIGS. 1 to 5: example 5 transmission electron microscopy photographs of indocyanine green fluorescently tagged trans-anethole-albumin nanoparticles were prepared.
FIGS. 1 to 6: example 6 transmission electron microscopy photographs of indocyanine green fluorescently tagged trans-anethole-albumin nanoparticles were prepared.
FIGS. 1 to 7: example 7 transmission electron microscopy photographs of making indocyanine green fluorescent-tagged trans-anethole-albumin nanoparticles.
FIGS. 1 to 8: example 8 transmission electron microscopy photographs of indocyanine green fluorescently tagged trans-anethole-albumin nanoparticles were prepared.
FIG. 2: graph of in vitro drug release versus time for indocyanine green fluorescently labeled trans-anethole-albumin nanoparticles.
FIG. 3: indocyanine green fluorescent-labeled trans-anethole-albumin nanoparticle cytotoxicity analysis histogram.
FIG. 4-1: fluorescence imaging for nuclear DAPI staining.
FIG. 4-2: trans-anethole-albumin nanoparticle endocytosis fluorescence imaging fluorescently tagged with indocyanine green.
FIGS. 4-3: the images are the imaging result images after the two fluorescence images Merge.
Detailed Description
The present invention will be further described with reference to specific examples, but the present invention is not limited thereto.
Example 1:
1) preparing medicine-carrying bovine serum albumin nanoparticles: taking 2ml of 10mg/ml bovine serum albumin aqueous solution, and carrying out ultrasonic treatment to make the solution uniform.
2) A solution of 40mg/ml trans-anethole 15. mu.l in dichloromethane and a solution of 80mg/ml indocyanine green 2. mu.l was added slowly with a pipette gun.
3) After stirring well, 100. mu.l of an aqueous solution of glutaraldehyde crosslinker was added rapidly, and the reaction was stirred at last 700rpm/min at 20 ℃ for 3 hours.
4) After the reaction was completed, the mixture was filtered through a 220nm aqueous membrane.
5) And centrifuging the filtrate at 8,000rpm/min for 4 minutes, and washing the filtrate for 5 times by using deionized water to obtain the trans-anethole-coated and green indole phthalocyanine bovine serum albumin nanoparticles.
Transmission electron microscopy of prepared indocyanine green fluorescent-tagged trans-anethole-albumin nanoparticles observed morphology (fig. 1-1).
Example 2:
1) preparing medicine-carrying bovine serum albumin nanoparticles: taking 2ml of 15mg/ml bovine serum albumin aqueous solution, and carrying out ultrasonic treatment to make the solution uniform.
2) 12 μ l of a 40mg/ml solution of trans-anethole in dichloromethane, and 1 μ l of an 80mg/ml solution of indocyanine green, were added slowly with a pipette gun.
3) After stirring well, 80. mu.l of an aqueous solution of glutaraldehyde crosslinker were quickly added and the reaction was stirred for 4 hours at the end at 800rpm/min at 20 ℃.
4) After the reaction was completed, the mixture was filtered through a 220nm aqueous membrane.
5) And centrifuging the filtrate at 8,000rpm/min for 4 minutes, and washing the filtrate for 5 times by using deionized water to obtain the trans-anethole-coated and green indole phthalocyanine bovine serum albumin nanoparticles.
Transmission electron microscopy of prepared indocyanine green fluorescent-tagged trans-anethole-albumin nanoparticles observed morphology (fig. 1-2).
Example 3:
1) preparing medicine-carrying bovine serum albumin nanoparticles: 2ml of 15mg/ml bovine serum albumin aqueous solution was taken and homogenized by sonication.
2) 12 μ l of a 50mg/ml solution of trans-anethole in dimethyl sulfoxide, and 1 μ l of a 90mg/ml solution of green indole phthalocyanine were added slowly with a pipette gun.
3) After stirring well, 100. mu.l of an aqueous solution of glutaraldehyde crosslinker was added rapidly, and the reaction was stirred at last 700rpm/min at 20 ℃ for 3 hours.
4) After the reaction was completed, the mixture was filtered through a 220nm aqueous membrane.
5) The filtrate was centrifuged at 7,000rpm/min for 5 minutes and washed 6 times with deionized water to obtain bovine serum albumin nanoparticles loaded with trans-anethole and green indole.
Transmission electron microscopy of prepared indocyanine green fluorescent-tagged trans-anethole-albumin nanoparticles observed morphology (fig. 1-3).
Example 4:
1) preparing medicine-carrying bovine serum albumin nanoparticles: 2ml of 20mg/ml bovine serum albumin aqueous solution was taken and homogenized by sonication.
2) A solution of 50mg/ml trans-anethole 15. mu.l in dimethyl sulfoxide, and a solution of 80mg/ml 2. mu.l of indocyanine green, were added slowly with a pipette gun.
3) After stirring well, 100. mu.l of an aqueous solution of glutaraldehyde crosslinker was added rapidly, and the reaction was stirred at last 700rpm/min at 20 ℃ for 3 hours.
4) After the reaction was completed, the mixture was filtered through a 220nm aqueous membrane.
5) And centrifuging the filtrate at 8,000rpm/min for 4 minutes, and washing the filtrate for 3 times by using deionized water to obtain the trans-anethole-coated and green indole phthalocyanine bovine serum albumin nanoparticles.
Transmission electron microscopy of prepared indocyanine green fluorescent-tagged trans-anethole-albumin nanoparticles observed morphology (fig. 1-4).
Example 5:
1) preparing medicine-carrying bovine serum albumin nanoparticles: taking 3ml of 10mg/ml bovine serum albumin aqueous solution, and carrying out ultrasonic treatment to make the solution uniform.
2) 10 μ l of a 60mg/ml solution of trans-anethole in ethanol, and 1.5 μ l of an 80mg/ml solution of indocyanine green, were added slowly with a pipette gun.
3) After stirring well, 70. mu.l of an aqueous solution of glutaraldehyde crosslinker were rapidly added and the reaction was stirred at last 700rpm/min at 20 ℃ for 3 hours.
4) After the reaction was completed, the mixture was filtered through a 220nm aqueous membrane.
5) The filtrate was centrifuged at 7,000rpm/min for 6 minutes and washed 3 times with deionized water to obtain bovine serum albumin nanoparticles loaded with trans-anethole and green indole.
Transmission electron microscopy of prepared indocyanine green fluorescent-tagged trans-anethole-albumin nanoparticles observed morphology (fig. 1-5).
Example 6:
1) preparing medicine-carrying bovine serum albumin nanoparticles: taking 3ml of 15mg/ml bovine serum albumin aqueous solution, and carrying out ultrasonic treatment to make the solution uniform.
2) Mu.l of a 50mg/ml solution of trans-anethole in ethanol and 3. mu.l of a 90mg/ml solution of indocyanine green were added slowly with a pipette gun.
3) After stirring well, 70. mu.l of an aqueous solution of glutaraldehyde crosslinker were rapidly added and the reaction was stirred for 2 hours at last 700rpm/min at 20 ℃.
4) After the reaction was completed, the mixture was filtered through a 220nm aqueous membrane.
5) And centrifuging the filtrate at 6,000rpm/min for 6 minutes, and washing the filtrate 4 times by deionized water to obtain the trans-anethole-coated and green indole phthalocyanine bovine serum albumin nanoparticles.
Transmission electron microscopy of prepared indocyanine green fluorescent-tagged trans-anethole-albumin nanoparticles observed morphology (fig. 1-6).
Example 7:
1) preparing drug-loaded human serum albumin nanoparticles: 2ml of 20mg/ml human serum albumin aqueous solution is taken and homogenized by ultrasonic treatment.
2) A solution of 40mg/ml trans-anethole 15. mu.l in dichloromethane and a solution of 80mg/ml indocyanine green 2. mu.l was added slowly with a pipette gun.
3) After stirring well, 100. mu.l of an aqueous solution of glutaraldehyde crosslinker was added rapidly, and the reaction was stirred at last 700rpm/min at 20 ℃ for 3 hours.
4) After the reaction was completed, the mixture was filtered through a 220nm aqueous membrane.
5) The filtrate was centrifuged at 8,000rpm/min for 4 minutes and washed 5 times with deionized water to obtain human serum albumin nanoparticles loaded with trans-anethole and green indole.
Transmission electron microscopy of prepared indocyanine green fluorescent-tagged trans-anethole-albumin nanoparticles observed morphology (fig. 1-7).
Example 8:
1) preparing drug-loaded human serum albumin nanoparticles: 2ml of 20mg/ml human serum albumin aqueous solution is taken and homogenized by ultrasonic treatment.
2) A solution of 40mg/ml trans-anethole 15. mu.l in dichloromethane and a solution of 80mg/ml indocyanine green 2. mu.l was added slowly with a pipette gun.
3) After stirring well, 100. mu.l of an aqueous solution of glutaraldehyde crosslinker was added rapidly, and the reaction was stirred at last 700rpm/min at 20 ℃ for 3 hours.
4) After the reaction was completed, the mixture was filtered through a 220nm aqueous membrane.
5) The filtrate was centrifuged at 8,000rpm/min for 4 minutes and washed 5 times with deionized water to obtain human serum albumin nanoparticles loaded with trans-anethole and green indole.
Transmission electron microscopy of prepared indocyanine green fluorescent-tagged trans-anethole-albumin nanoparticles observed morphology (fig. 1-8).
From the examples, we have successfully prepared indocyanine green trans-anethole-albumin nanoparticles with good sphericity, uniform particles and diameters of 20-50 nanometers.
Example 9
1) The trans-anethole-albumin nanoparticle stock solution prepared by the method is divided into a plurality of parts by taking a phosphate buffer solution with pH of 7.4 as a drug release medium, and is shaken at a constant speed at 37 ℃ on a shaking table, and the samples are sampled at fixed time to perform in-vitro slow release experiments. The mixture was centrifuged at 8,000rpm/min for 4 minutes in a 100K ultrafiltration tube, and the resulting centrifugate was collected.
2) The in-vitro release rule of the trans-anethole-albumin nano particles is inspected by adopting a high performance liquid chromatography to calculate the release rate.
Taking 100 mu l of stock solution of anethole particles, adding 50 mu l of DMSO to fully dissolve the released anethole, and performing ultrasonic treatment by an ultrasonic probe for 3 minutes to fully break the particles to release the encapsulated anethole. The high performance liquid chromatography is used for measuring the absorption peak area of trans-anethole in the particle stock solution and the absorption peak area of anethole in the sustained-release centrifugate, and 50 mu l of the sample is loaded each time. The drug release rate of the drug is obtained according to the relation of the peak area ratio of the drug absorption
Absorption peak area S of stock solution0And the absorption peak area of the centrifugate slowly released every day is recorded as S1、S2、S3、S4、S5。
Absorption peak area S of stock solution0380.79 (per 50. mu.l), S: (1...5) The absorption peak area was 9.9-43.5 (per 50. mu.l), and the calculated drug release rates were 2.6%, 4.2%, 4.5%, 11.65%, and 31%, respectively, as a sustained release curve (FIG. 2).
Example 10
MTT assay for cytotoxicity of trans-anethole-albumin nanoparticles:
1) mouse mononuclear macrophage in logarithmic growth phase is digested with 0.125% trypsin and blown into single cell suspension, and the cell concentration is regulated to 5 × 104/m1 after counting.
2) Mu.l of the cell suspension was added to each well of a 96-well plate and cultured at 37 ℃ for 24 hours in a 5% CO2 incubator.
3) Mu.l of the trans-anethole-albumin nanoparticle solutions containing different concentrations (0.03mg/ml, 0.01mg/ml, 0.003mg/ml, 0.001mg/ml, 0.0003mg/ml, 0.0001mg/ml) were added to fresh medium and CO-cultured for 24h with cells in a 5% CO2 incubator at 37 ℃.
4) After 24h the plates were removed and 10. mu.l MTT solution was added to each well and after further incubation for 4h at 37 ℃ in a 5% CO2 incubator, the medium was carefully aspirated from the wells and 200. mu.l dimethyl sulfoxide (DMSO) solution was added to each well and shaken on a shaker for 20min to allow the DMSO solution to fully dissolve the Formazan crystals.
5) The cell viability was calculated by zeroing the blank wells with an enzyme-linked immunosorbent assay at a wavelength of 570nm, measuring the absorbance A of each well, and using the following formula. Each group was set to 8 replicates and the average was calculated.
Cell viability (%). test cell set absorbance (a 2)/blank cell set absorbance (a 1). 100%
The blank group light absorption value is 0.6368, the experimental group cell light absorption value is 0.4805-0.5993, and the calculated cell survival rates are 75.45%, 86.90%, 88.69%, 93%, 93.79% and 94% respectively. A bar graph of the trans-anethole-albumin nanoparticle cytotoxicity assay was made based on the calculated viability for the different concentration cell groups (figure 3).
Example 11
Trans-anethole-albumin nanoparticle endocytosis experiments were performed in mouse mononuclear macrophages:
1) the culture medium of mouse mononuclear macrophages with a 24-well plate or 48-well plate was changed to a serum-free medium, and starvation-cultured for 2 hours at 37 ℃ in a 5% carbon dioxide incubator.
2) Trans-anethole-albumin nanoparticles containing green indocyanine dye were then added to mouse mononuclear macrophages, and the medium was aspirated away after 4 hours of culture.
3) Cells were washed 3 times with PBS, then fixed with 4% paraformaldehyde for 10min, and washed 3 times with PBS. Staining the cell nucleus with 0.5 mu g/ml DAPI staining solution, washing with PBS for three times after 10min, observing by a fluorescence microscope, and taking a trans-anethole-albumin nanoparticle endocytosis fluorescence imaging picture. FIG. 4-1 is fluorescence imaging of DAPI staining of cell nuclei, FIG. 4-2 is fluorescence imaging of trans-anethole-albumin nanoparticle endocytosis, and FIG. 4-3 is an imaging result picture after two fluorescence pictures Merge.
From the fluorescence imaging result of endocytosis, a great amount of trans-anethole-albumin nanoparticles are endocytosed into mouse mononuclear macrophages, and the imaging tracing function of the trans-anethole-albumin nanoparticles is successfully characterized.
According to the preparation method of the trans-anethole-albumin nano-particles labeled by green indocyanine green fluorescence, disclosed and provided by the invention, a person skilled in the art can realize the preparation of the particles by referring to the content in the text and appropriately changing links such as condition routes and the like. Although the method and the preparation technique of the present invention have been studied in the past, there has been no report on albumin particles encapsulating both trans-anethole and a fluorescent dye. It will be apparent to those skilled in the art that modifications and rearrangements of the methods and technical routines described herein can be made to achieve the desired end result without departing from the spirit, scope, and spirit of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and content of the invention.
Claims (5)
1. A preparation method of indocyanine green fluorescent-labeled trans-anethole-albumin nanoparticles; the method is characterized by comprising the following steps:
1) preparing drug-loaded albumin nanoparticles: preparing 2-2.5 ml of 10-20 mg/ml albumin aqueous solution;
2) adding 10-15 mul of trans-anethole solution with the concentration of 40-60 mg/ml prepared by dichloromethane, ethanol or dimethyl sulfoxide and 1-3 mul of green indole phthalocyanine solution with the concentration of 80-100 mg/ml;
3) stirring and reacting at the temperature of 5-50 ℃ at 600-800 rpm/min for 2-6 hours, adding 70-100 mu l of glutaraldehyde crosslinking agent aqueous solution after stirring, and stirring and reacting at the temperature of 5-50 ℃;
4) after the reaction is finished, filtering by using a water system filter membrane;
5) and (3) performing centrifugal separation on the filtrate, and washing with deionized water to obtain the trans-anethole-albumin nano-particles with diameters of 20-50 nanometers and drug loading rates of 5-8% and fluorescent tracing of green indocyanine.
2. The method as set forth in claim 1, wherein the step 4) is performed by filtration through a 220nm water-based filter.
3. The method as set forth in claim 1, wherein the filtrate of step 5) is centrifuged at 6000 to 8,000rpm/min for 3 to 6 minutes.
4. The method according to claim 1, wherein the deionized water is washed 3 to 6 times in the step 5).
5. The method of claim 1, wherein the albumin is bovine serum albumin or human serum albumin.
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